Nucleic acid amplification and testing

ABSTRACT

Methods for amplifying a target nucleic acid by self-sustained amplification methods are described. The methods are designed, in particular, to be carried out without use of specialised lab facilities or instruments. Compositions, lyophilised formulations, and kits for carrying out the methods are also described.

This invention relates to methods for amplifying a target nucleic acid,and to methods for nucleic acid testing, particularly methods that canbe carried out without specialised lab facilities or instruments.Compositions, formulations, and kits for nucleic acid amplification arealso provided, as well as lyophilised formulations and kits comprisinglyophilised formulations.

Nucleic acid testing is used for many purposes such as screening anddiagnosis of infectious diseases and genetic disorders, testing fordisease susceptibility, monitoring progression of treatment, andimproving the safety of blood supplies. Nucleic acid testing combinesthe advantages of direct and highly sequence-specific detection ofnucleic acid of an infectious agent with an analytic sensitivity that isseveral orders of magnitude higher than that of immuno-based tests, orvirus isolation and cell culture methods. Nucleic acid testing alsoreduces the risk of infectious agent transmission between infection andseroconversion, of infection with immunovariant viruses, and ofimmunosilent or occult carriage.

Well known methods of nucleic acid testing involve use of reversetranscription of RNA followed by PCR(RT-PCR) to amplify RNA species.However, RT-PCR suffers from the disadvantage that it involves repeatedwide changes in sample temperature, for which specialised thermalcycling instruments are required. A further disadvantage is thatamplified RNA template can be difficult to differentiate from amplifiedcontaminating double stranded DNA.

An alternative RNA amplification strategy to RT-PCR is termedtranscription-based amplification. Such methods involve amplification ofan RNA template using reverse transcriptase (RT), RNase H, and RNApolymerase activities, and include nucleic acid sequence-basedamplification (NASBA), transcription-mediated amplification (TMA), andself-sustained sequence replication (3SR) (Chan and Fox, Rev. Med.Microbiol. 10: 185-196 (1999); Guatelli et al., Proc. Natl. Acad. Sci.87: 1874-1878 (1990); Compton, Nature 350:91-92 (1991)). NASBA and 3SRuse RT from Avian Myeloblastosis Virus (AMV) (which also has RNaseHactivity), RNase H from E. coli, and T7 RNA polymerase. TMA uses MoloneyMurine Leukemia Virus (MMLV) RT (which also has RNase H activity), andT7 RNA polymerase.

Transcription-based amplification methods have several advantages overRT-PCR. The reactions occur simultaneously in a single tube, and arecarried out under isothermal conditions so a thermocycler is notrequired. The amplification reaction is faster than RT-PCR (1×10⁹-foldamplification can be seen after five cycles, compared with 1×10⁶-foldamplification after 20 cycles for RT-PCR). DNA background does notinterfere with transcription-based amplification, and so these methodsare not affected by double stranded DNA contamination. The amplificationproduct is single stranded and can be detected without any requirementfor strand separation.

Conventional transcription-based amplification methods, however, sufferfrom the disadvantage that they have lower specificity than RT-PCR. Itis also necessary to denature the nucleic acid template by heating thesample, and then cooling before adding the enzymes required foramplification of the template, thereby increasing the complexity of themethod. Transcription-based amplification methods are also not as robustas RT-PCR. Conventional NASBA is sensitive to temperature fluctuationsexceeding +/−0.5° C.

U.S. Pat. No. 5,981,183 describes a method of transcription-basedamplification which uses thermostable enzymes. The amplificationreaction is carried out at 50-70° C., thereby improving specificity.However, a disadvantage of this method is that it is still susceptibleto false positives caused by false priming of denatured double strandedDNA. Such methods are also not well suited to use in the field becauseof the need to heat the reaction to 50-70° C.

A further disadvantage of conventional nucleic acid testing is thatdetection of the amplified reaction product requires time-consuming,labour-intensive electrophoretic separation of the reaction products, orexpensive equipment to detect fluorescent or chemiluminescent signals.The reagents required are expensive and must be transported and storedbelow room temperature. Separate designated areas are required for thesample preparation, amplification, and detection steps of the methods.The methods can only be carried out in specialized, well-equippedlaboratories, by highly trained technicians. Consequently, conventionalmethods are not suitable for near-patient or field testing, and areunaffordable in poorer regions with a high prevalence of infectiousdisease (such as Africa, Asia, and Latin America) where they are mostneeded.

There is, therefore, a need to provide methods of nucleic acid testingthat can be carried out without use of specialised lab facilities orinstruments, and which have high specificity for target nucleic acid.

Lyophilisation has been used to store enzymes for nucleic acidamplification reactions. U.S. Pat. No. 5,556,771 describes lyophilisedformulations that comprise MMLV RT and T7 polymerase with trehalose andpolyvinyl pyrrolidine as cryoprotectant stabilizing agents. However, theresults described in U.S. Pat. No. 5,556,771 show some loss in activity(measured as the ability to cause nucleic acid amplification) afterstorage at 35° C. for 61 days. Instructions provided with commerciallyavailable kits comprising lyophilised reagents for carrying outtranscription-mediated amplification (GEN-PROBE® APTIMA® General PurposeReagents (GPR) 250 Kit) or PCR amplification (SmartMix™ HM of Cepheid)require the lyophilised reagents to be stored at 2-8° C. Instructionsprovided with a commercially available kit containing lyophilisedreagents for NASBA-based nucleic acid amplification (Nuclisens® BasicKit Amplification Reagents of Biomérieux) specifies that theamplification reagents should be stored at ≤−20° C. We have also foundthat lyophilised formulations disclosed in U.S. Pat. No. 5,556,771 andin the above commercially available kits do not reconstitute rapidly,but instead require extensive mixing in specially formulatedreconstitution buffers.

There is, therefore, a need to provide lyophilised formulations that canpreserve labile reagents in a stable condition for long periods atambient temperature, and which can be easily and rapidly rehydrated.

According to the invention there is provided a method of nucleic acidamplification, which comprises amplifying a target nucleic acid by aself-sustained amplification reaction which is carried out at atemperature between 42° C. and 50° C.

The term “self-sustained amplification reaction” is used herein toinclude nucleic acid amplification reactions in which copies of a targetnucleic acid are produced, which then function as templates forproduction of further copies of target nucleic acid (either sense oranti-sense copies). The reactions are self-sustained reactions that canoccur under isothermal conditions, and so there is no requirement forthermal cycling during the amplification reaction (unlike, for example,the polymerase chain reaction (PCR)). Examples of self-sustainedamplification reactions are known to the skilled person, and includetranscription-based amplifications, strand displacement amplification(SDA), rolling-circle amplification (RCA), Q beta replicaseamplification, and loop-mediated isothermal amplification (LAMP).

It has been found that the time taken to obtain a desired copy number ofamplification product using a method of the invention is surprisinglymuch less than the time taken to achieve the same copy number withconventional self-sustained amplification methods. In our experience,methods of the invention are approximately twice as quick asconventional methods. A suitable incubation time at between 42° C. and50° C. is at least 30 minutes, or at least 40 minutes. 45-55 minutes maybe optimal.

Whilst the temperature may vary between 42° C. and 50° C. when carryingout the amplification reaction, it is expected that the amplificationreaction will generally be carried out under substantially isothermalconditions, i.e. within a temperature range of 1-3° C. A suitable way ofachieving this is to incubate the amplification reaction using a waterbath.

The phrase “at a temperature between 42° C. and 50° C.” means above 42°C. and less than 50° C. The self-sustained amplification reaction may becarried out at a temperature above 43° C. and less than 50° C. Theself-sustained amplification reaction may be carried out at atemperature of 43-49° C. The reaction may be carried out at atemperature of 43-49° C., 44-49° C., 45-49° C., or 45-48° C.

The target nucleic acid may be DNA (single or double stranded) or RNA.The target nucleic acid may be any target nucleic acid that it isdesired to amplify or detect, including ribosomal RNA, viral orbacterial RNA or DNA. The target nucleic acid may be nucleic acid of (orderived from) a disease causing micro-organism (for example HCV). Thetarget nucleic acid may be nucleic acid of an organism associated with asexually transmitted disease, such as Chlamydia trachomatis, or HIV. Inother embodiments, the target nucleic acid may be nucleic acid of asubject (for example to determine a particular genotype of the subject).

Methods of the invention may be used to determine whether or not atarget nucleic acid is present in a sample solution suspected ofcontaining the target nucleic acid. Accordingly, there is also providedaccording to the invention a method of testing for the presence of atarget nucleic acid in a sample solution suspected of containing thetarget nucleic acid, the method comprising incubating the samplesolution under conditions for amplification of the target nucleic acidby a self-sustained amplification reaction at a temperature between 42°C. and 50° C.

The sample solution may be any solution suspected of containing a targetnucleic acid which it is desired to detect. The sample solution may be,or be derived from, a biological sample obtained from a subject.Examples of biological samples are blood, serum, urine, a cervicalsmear, a swab sample, or tissue homogenate.

It will be appreciated that it may be necessary to extract nucleic acidfrom the biological sample to provide a sample solution that can betested in accordance with the invention to determine whether or not atarget nucleic acid is present. Nucleic acid extraction may be carriedout using any suitable nucleic acid extraction method. Suitable methodsinclude solid phase extraction of nucleic acid on silica particles (Boomet al., J. Clin. Microbiol; 28:495-503, 1990) or silica gel/glass fibrefilters (Vogelstein et al., Proc. Natl. Acad. Sci., 76: 615-619, 1979)in the presence of chaotropic salts, using commercially available kits(for example from Qiagen, Roche, or Invitrogen). Alternative methodsinclude: liquid phase extraction technology based in acid guanidiniumthiocyanate-phenol-chloroform extraction (Chomczynski et al., AnalBiochemistry; 162: 156-159, 1987); FTA® kit protocol by Whatmann usingFTA® paper filter matrixes impregnated by chemical formula that lysescell membranes and immobilizes the nucleic acids; Charge Switch™technology (Baker et al., EP1036082). Alternative, more simpleprocedures involve sample lysis by heat or chemical treatment and sampledilution prior to amplification.

Methods of the invention for determining whether or not a target nucleicacid is present in a sample solution may be used to test a biologicalsample obtained from a subject to see whether the subject is infectedwith an infectious agent, or to monitor the subject for progression of adisease, or for response to treatment.

Examples of infectious agents include bacterial or viral infectiousagents, such as HIV, HCV, HPV, CMV, HTLV, EBV, rhinovirus, measlesvirus. Infectious agents may be those associated with sexuallytransmitted disease, such as Chlamydia trachomatis, or HIV.

Suitable self-sustained nucleic acid amplification reactions may becarried out using the following enzyme activities: an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specificribonuclease, and a DNA-dependent RNA polymerase.

The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, andDNA/RNA duplex-specific ribonuclease activities may be provided by asingle enzyme (for example AMV-RT or MMLV-RT). Additional DNA/RNAduplex-specific ribonuclease activity may be provided by a separateenzyme (for example an RNaseH). In some embodiments, AMV-RT and RNaseHmay be used (as in NASBA).

As explained above, self-sustained nucleic acid amplification reactionsare known to the skilled person. Suitable types of reaction that may beused in accordance with the invention include transcription-basedamplification methods, such as methods corresponding to NASBA, TMA, or3SR (i.e. methods which are the same as conventional NASBA, TMA, or 3SR,but carried out at a temperature between 42° C. and 50° C.).

A transcription-based self-sustained amplification reaction suitable foruse in methods of the invention is described below, with reference toFIG. 1.

An antisense Primer 1 comprises nucleic acid sequence complementary to aportion of a target RNA so that the primer can hybridise specifically tothe target RNA, and a single stranded-version of a promoter sequence fora DNA-dependent RNA polymerase at its 5′-end. Primer 1 is annealed tothe RNA target. An RNA-dependent DNA polymerase extends Primer 1 tosynthesise a complementary DNA (cDNA) copy of the RNA target. A DNA/RNAduplex-specific ribonuclease digests the RNA of the RNA-cDNA hybrid. Asense Primer 2 comprises nucleic acid sequence complementary to aportion of the cDNA. Primer 2 is annealed to the cDNA downstream of thepart of the cDNA formed by Primer 1. Primer 2 is extended by aDNA-dependent DNA polymerase to produce a second DNA strand whichextends through the DNA-dependent RNA polymerase promoter sequence atone end (thereby forming a double stranded promoter). This promoter isused by a DNA-dependent RNA polymerase to synthesise a large number ofRNAs complementary to the original target sequence. These RNA productsthen function as templates for a cyclic phase of the reaction, but withthe primer annealing steps reversed, i.e., Primer 2 followed by Primer1.

In a variation of this method, Primer 2 may also include a singlestranded version of a promoter sequence for the DNA-dependent RNApolymerase. This results in production of RNAs with the same sense asthe original target sequence (as well as RNAs complementary to theoriginal target sequence).

In some conventional self-sustained transcription-based amplificationreactions it is known to cleave the target RNA at the 5′-end before itserves as the template for cDNA synthesis. An enzyme with RNase Hactivity is used to cleave the RNA portion of an RNA-DNA hybrid formedby adding an oligonucleotide (a cleavage oligonucleotide) having asequence complementary to the region overlapping and adjacent to the5′-end of the target RNA. The cleavage oligonucleotide may have its3′-terminal-OH appropriately modified to prevent extension reaction.Whilst in some embodiments of the invention a cleavage oligonucleotidecould be used, it is preferred that a method of the invention is carriedout in the absence of a cleavage oligonucleotide thereby simplifying theamplification reaction and the components required.

It will be appreciated that in addition to the required enzymeactivities, it will also be necessary to provide appropriate nucleotidetriphosphates (for transcription-based amplifications ribonucleotidetriphosphates (rNTPs, i.e. rATP, rGTP, rCTP, and rUTP), anddeoxyribonucleotide triphosphates (dNTPs, i.e. dATP, dGTP, dCTP, anddTTP) are required), appropriate primers for specific amplification ofthe target nucleic acid, a suitable buffer for carrying out theamplification reaction, and any necessary cofactors (for examplemagnesium ions) required by the enzyme activities. Examples of suitablebuffers include Tris-HCl, HEPES, or acetate buffer.

Accordingly, conditions for amplification of the target nucleic acidused in methods of the invention for testing for the presence of atarget nucleic acid in a sample solution may comprise enzyme activitiesrequired for the self-sustained amplification reaction (for example,RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, DNA/RNAduplex-specific ribonuclease, and DNA-dependent RNA polymerase enzymeactivities), cofactors required by the enzyme activities (for examplemagnesium ions), primers for specific amplification of the targetnucleic acid, appropriate nucleotide triphosphates (ribonucleotidetriphosphates and deoxyribonucleotide triphosphates are required fortranscription-based amplifications). Conditions for amplification of thetarget nucleic acid should also include a suitable buffer (such asTris-HCl, HEPES, or acetate buffer). A suitable salt may be provided,such as potassium chloride or sodium chloride.

Suitable concentrations of these components may readily be determined bythe skilled person. We have found that suitable rNTP concentrations aretypically in the range 0.25-5 mM, preferably 0.5-2.5 mM. Suitable dNTPconcentrations are typically in the range 0.25-5 mM dNTP, preferably0.5-2.5 mM. Suitable magnesium ion concentrations are typically in therange 5-15 mM.

Whilst it is possible that the self-sustained amplification reaction maybe carried out in the temperature range of between 42° C. and 50° C.using thermostable enzymes, the Applicant has appreciated thatnon-thermostable enzymes may be used in this temperature range (providedthe non-thermostable enzyme retains activity between 42 and 50° C.). Theterm “thermostable enzyme” is used herein to mean an enzyme with optimalenzymatic activity at 50° C. or above. Typically a thermostable enzymemaintains its activity at temperatures at least in excess of 55° C. andup to about 72° C. or higher. A “non-thermostable enzyme” has optimalenzymatic activity below 50° C., suitably in the temperature range of37-41° C. (although the non-thermostable enzyme may still retainactivity at 50° C. or above). Accordingly, in certain embodiments of theinvention, at least one of the enzyme activities (for example theDNA-dependent RNA polymerase activity) is provided by a non-thermostableenzyme. In some embodiments, all of the enzyme activities use for theamplification reaction may be provided by non-thermostable enzymes. Useof non-thermostable enzymes is preferred because this allows theamplification reaction to proceed efficiently at a temperature between42° C. and 50° C. Thermostable enzymes generally have optimum activityat temperatures above this range.

Some conventional transcription-based amplification methods use veryhigh amounts of T7 RNA polymerase (for example 142 or more units, whereone unit incorporates 1 nmole of labelled nucleotide into acid insolublematerial in 1 hour at 37° C. under standard assay conditions, such as:40 mM Tris-HCl (pH8.0), 50 mM NaCl, 8 mM MgCl₂, 5 mM DTT, 400 μM rNTPs,400 μM [³H]-UTP(30 cpm/pmoles), 20 μg/ml T7 DNA, 50 μg/ml BSA, 100 μlreaction volume, 37° C., 10 min.). We have found that methods of theinvention can be carried out using significantly less T7 RNA polymerasethan such conventional methods, thereby reducing cost. Thus, methods ofthe invention are preferably carried out using less than 142 units of aDNA-dependent RNA polymerase (for example T7 RNA polymerase), suitablyless than 100 units or less than 50 units, such as 30-40 units.

It may be desirable to include one or more agents that may facilitate orenhance the self-sustained amplification reaction at temperaturesbetween 42° C. and 50° C. An agent may facilitate or enhance thereaction by any mechanism, but typically the agent is not essential forthe reaction to take place, and may not directly take part in thereaction. Some such agents may act by helping to stabilize the activityof an enzyme required for the self-sustained amplification reactionbetween 42° C. and 50° C., or by reducing the effect of any inhibitorsof the self-sustained amplification reaction that may be present.

Examples of suitable agents include the following:

-   -   i) an inert protein. The term “inert protein” is used herein to        mean a protein which does not take part in the amplification        reaction. Suitable examples include bovine serum albumin (BSA),        casein, gelatin, or lysozyme. A suitable concentration range of        the inert protein is 0.01-1 μg/μl. A The protein should be        RNase-free;    -   ii) a reducing agent, such as Dithiothreitol (DTT) (for example        at a concentration of 1-5 mM) or n-acetylcysteine (NAC) (for        example at a concentration of 0.1-1M);    -   iii) an inert amphiphilic polymer (which is not a protein). The        term “inert amphiphilic polymer” is used herein to mean an        amphiphilic polymer which does not take part in the        amplification reaction. The inert amphiphilic polymer may be        non-charged. Examples of inert amphiphilic polymers are        polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) or        other similar polyole. A suitable concentration range of the        inert amphiphilic polymer is 0.1-2%;    -   iv) a sugar-alcohol, for example sorbitol, mannitol, or glycerol        (for example up to 5% glycerol). A suitable concentration range        is 0.1-2 M;    -   v) a low molecular weight saccharide, suitably a monosaccharide,        disaccharide, or trisaccharide. Examples of disaccharides are        trehalose, sucrose and maltose. A suitable concentration range        is 2.5%-15%;    -   vi) homopolymeric nucleic acid (200-2000 bases). Examples        include: Poly A, Poly C, or Poly U nucleic acid (for example at        100-600 ng/amplification); poly dA, poly dC or poly dI (for        example at 100-600 ng/amplification); tRNA; rRNA;    -   vii) acetate salts, for example magnesium or potassium acetate;    -   viii) spermidine, for example at 0.5-3 mM;    -   ix) poly-Lysine, for example at 0.2-3 mM;    -   x) detergent, for example NP40 or Tween20 (suitably at        0.01-0.5%).

The concentration of each of the above agents may be optimised for eachdifferent target nucleic acid and set of primers used for amplification.

Other agents may act by facilitating primer annealing or facilitatingdenaturation of double stranded nucleic acid. Accordingly, it may bedesired alternatively or additionally to include in the amplificationreaction one or more agents that facilitate primer annealing and/or oneor more agents that facilitate denaturation of double stranded nucleicacid.

Examples of agents that facilitate primer annealing are positivelycharged ions, such as potassium or sodium ions. Potassium ion may beprovided by potassium chloride or acetate (suitably at a concentrationof 30-200 mM or 30-90 mM). Sodium ion may be provided by sodium chlorideor acetate (suitably at a concentration of 50-400 mM).

Examples of agents that facilitate denaturation of double strandednucleic acid are:

-   i) polar aprotic solvents, such as dimethyl sulfoxide (DMSO), for    example at a concentration of 3-20% (v/v), or less than 10% (v/v).    It has been found that when nucleic acid amplification is carried    out in the temperature range of between 42 and 50° C., the amount of    polar aprotic solvent required to have an effect is less than the    amount required at lower temperatures. Alternative polar aprotic    solvents that may be used include tetramethylene sulfone, or    tetramethylene sulfoxide;-   ii) a zwitterionic compound, such as betaine    (N,N,N-trimethylglycine), for example at a concentration of 0.2-3M.    Betaine may be used in place of DMSO. An advantage of betaine is    that it is more stable than DMSO, and it does not appear to inhibit    lyophilisation. Consequently, use of betaine instead of DMSO may be    suitable where lyophilised reagents are used for the amplification    reaction. In some circumstances, however, use of betaine and DMSO    may be desired since synergistic effects of these reagents on double    stranded nucleic acid denaturation have been observed. Alternative    zwitterionic compounds that may be used include monomethylglycine,    dimethylglycine, D-carnitine, homoectoine, L-ectoine or derivatives;-   iii) a modified nucleotide triphosphate (NTP) that comprises a base    that can base pair with guanine or cytosine such that the melting    temperature of the base pair is less than the melting temperature of    a guanine-cytosine base pair. An example of a modified NTP is    inosine triphosphate (rITP). An inosine-cytosine (I-C) base pair    comprises two hydrogen bonds between the bases, compared with a    guanine-cytosine (G-C) base pair which comprises three hydrogen    bonds, and so the melting temperature of the I-C base pair is less    than a G-C base pair. For example, rITP may be present in the    amplification reaction at a concentration of 0.5-4 mM or 0.5-3.5 mM.    When rITP is present, the amount of rGTP in the amplification    reaction may be reduced compared to the amounts of the other    ribonucleotide triphosphates (rATP, rCTP, rUTP) to compensate for    the amount of rITP. The ratio of rITP:rGTP will depend on the target    nucleic acid and on the primers that are used. A typical ratio is    1:3 to 1:1.5 rITP:rGTP;-   iv) a single-stranded nucleic acid binding protein, such as    Single-stranded Binding Protein (SSB), or Phage T4 gene 32 protein.    SSB and Phage T4 gene 32 protein bind single stranded regions of DNA    and thereby inhibit formation of double stranded DNA or DNA/RNA    hybrids.

Other agents may improve the specificity of the amplification reaction.Accordingly, it may be desired alternatively or additionally to includeone or more such agents in the amplification reaction.

Examples of agents that may improve the specificity of the amplificationreaction are:

-   i) ammonium ions, such as tetramethylammonium chloride (TMAC).    Ammonium ions are thought to interfere with week hydrogen bond    formation and create more stringent and specific hybridisation    conditions;-   ii) EDTA, EGTA, nitroacetic acid (NTA), uramil diacetic acid (UDA),    trans-1,2-cyclohexanediaminetetraacetic acid (CyDTA),    diethylenetriaminepentaacetic acid (DPTA),    ethyleneglycolbis(2-aminoethyl)ether diaminetetraacetic acid    (GEDTA), triethylenetetraminehexaacetic acid (TTHA), or a salt    thereof. These compounds are believed to improve the signal-to-noise    ratio of amplification reactions by significantly inhibiting the    occurrence of non-specific amplification reactions;-   iii) carrier nucleic acid with one or more magnesium salts. This is    believed to reduce polymerase extension of non-target nucleic acids    during amplification through a reduction in the amount of    primer-dimer formation.

In conventional transcription-based amplification methods, it isgenerally necessary to carry out an initial incubation of samplecontaining the target nucleic acid at a raised temperature beforecooling the sample. This reduces secondary structure in the targetnucleic acid, and allows primers for use in the amplification reactionto anneal to the target nucleic acid. Unless enzymes are used thatretain activity after incubation at the raised temperature, this initialincubation step is carried out before addition of the enzymes requiredfor the amplification reaction.

It has been found, however, that carrying out the self-sustainedamplification reaction at a temperature between 42° C. and 50° C. doesnot require a preliminary raised temperature incubation step to becarried out. Consequently, methods of the invention are simplifiedcompared to conventional transcription-based amplification methods, andcan be carried out more rapidly than such methods. These areparticularly important advantages for nucleic acid amplificationreactions carried out in the field.

Of course, in some circumstances, it may nonetheless be desirable tocarry out a raised temperature pre-incubation step. If the targetnucleic acid is double stranded DNA, it will usually be necessary todenature the target before carrying out the amplification reaction.Double stranded DNA may be denatured by chemical methods well known tothe skilled person, or alternatively by raised temperature denaturation.

Once the amplification reaction has been carried out, it will usually bedesired to detect product of the amplification reaction (referred to as“amplification product” below). Single or double stranded amplificationproduct may be detected. For example, double stranded amplificationproduct produced during the cyclic phase of the self-sustainedamplification reaction illustrated in FIG. 1 and described above may bedetected. If single stranded amplification product is detected thisremoves any requirement for separation of double strands, and thereforesimplifies detection.

Detection of amplification product may be carried out using any suitablemethod. For example, an instrument-independent detection method may beused, for example allowing visual detection of the amplificationproduct.

According to particular embodiments of the invention amplificationproduct may be detected using a dipstick. In suitable methods ofdipstick detection, amplification product is transported along adipstick by capillary action to a capture zone of the dipstick, anddetected at the capture zone. Amplification product may be captured anddetected using a sandwich nucleic acid dipstick detection assay in whichthe amplification product is immobilised at the capture zone of thedipstick by hybridisation to a capture probe, and detected at thecapture zone by hybridisation to a detection probe.

Methods of detection of nucleic acid by dipstick assay are known to theskilled person. The Applicant has developed particularly sensitivemethods of dipstick detection, which are described in WO 02/004667, WO02/04668, WO 02/004669, WO 02/04671, and in Dineva et al (Journal ofClinical Microbiology, 2005, Vol. 43(8): 4015-4021).

It is well known that a disadvantage of conventional nucleic acidamplification reactions is the risk of contamination of target nucleicacid with non-target nucleic acid that can lead to false positives.Conventionally, the risk of contamination in nucleic acid amplificationreactions is minimised by carrying out the reactions in laboratoriesusing separate dedicated areas for sample preparation, nucleic acidamplification, and detection of amplified nucleic acid. It will beappreciated, however, that this is not possible when nucleic acidamplification reactions are carried out away from such facilities (forexample in the field, in a physician's office, at home, in remote areas,or in developing countries where specialist facilities may not beavailable).

The Applicant has appreciated that when a nucleic acid amplificationreaction is carried out away from specialised lab facilities, risk ofcontamination can be reduced by performing the amplification reaction ina processing chamber that is sealed from the external environment. Thenucleic acid amplification reaction may be carried out in accordancewith a method of the invention. Detection of the amplification productmay then be carried out in an analysing chamber that is also sealed fromthe external environment.

The processing chamber and analysing chamber may be provided by adevice. The device may be preloaded with reagents (preferably inlyophilised form) required for amplification of the target nucleic acid(including enzyme activities) and/or detection of the amplificationproduct.

The risk of contamination of other samples with amplification productcan be reduced by treatment of the amplification product with nucleicacid modifying or hydrolysing agents that prevent its furtheramplification. A suitable treatment is chemical treatment that modifiesand degrades nucleic acid, for example non-enzymatic degradation ofnucleic acid by chemical nucleases. Examples of chemical nucleases aredivalent metal chelate complexes, such as copper Phenantroline-Cu (II)or Ascorbate-Cu (II) cleavage, as described by Sigman et al (J. Biol.Chem. (1979) 254, 12269-12272) and Chiou (J. Biochem (1984) 96,1307-1310). Alternatively, a base that is not naturally present in thetarget nucleic acid can be incorporated into the amplification product.For example, dUTP can be used to incorporate uracil into a DNAamplification product (as described in U.S. Pat. No. 5,035,996). If,prior to amplification, uracil DNA glycosylase (UDG) is then added to asample that may have been contaminated with such DNA amplificationproduct this will cause enzymatic hydrolysis of any contaminatingamplification product (containing uracil) without affecting natural DNAin the sample.

Reagents required for amplification of the target nucleic acid and/ordetection of the amplification product may be provided in lyophilisedform. Lyophilisation improves the stability of the reagents, therebyallowing them to be stored for longer periods at higher temperatures.Lyophilisation also reduces the weight and volume of the reagents sothat they are easier to transport. Use of lyophilised reagents is,therefore, advantageous for carrying out methods of the invention in thefield.

The Applicant has developed lyophilisation formulations (i.e.formulations suitable for lyophilisation) which (once lyophilised) areable to maintain reagents in a stable condition at temperatures up to37° C. for at least a year. This removes any requirement for coldstorage or cold-chain transport of the reagents. The formulations alsohave the advantage that they can be rapidly rehydrated afterlyophilisation. This is a particularly desirable property of lyophilisedformulations used for nucleic acid testing in the field since the speedor accuracy of a test can be adversely affected if a reagent requiredfor amplification of a nucleic acid target or detection of amplificationproduct is not rehydrated readily during the amplification or detectionmethod.

According to the invention there is provided a lyophilisationformulation comprising a polysaccharide, a low molecular weightsaccharide, and optionally a labile reagent which it is desired topreserve in a stable condition.

The term “labile reagent” is used herein to include any reagent that issusceptible to alteration or degradation when stored in aqueous solutionat ambient temperature. Examples of labile reagents include:biomolecules, such as proteins, peptides, or nucleic acids, orderivatives thereof, or chemicals such as enzyme cofactors, enzymesubstrates, nucleotide triphosphates (ribo- or deoxyribo-nucleotidetriphosphates), or salts. Examples of proteins include enzymes (forexample polymerases, such as DNA or RNA polymerases), and antibodies(native or recombinant, and fragments or derivatives thereof that retainantigen binding activity). An antibody (or fragment or derivative) maybe present in the formulation in the absence of an antigen bound by theantibody. Examples of nucleic acids include DNA, RNA, nucleic acidprimers, and carrier nucleic acid.

The labile reagent may be an amplification reagent for amplifying atarget nucleic acid (for example, by a self-sustained amplificationreaction), or a detection reagent for detecting product resulting fromamplification of target nucleic acid.

The amplification reagent may be any reagent required for amplificationof a target nucleic acid. For example the amplification reagent maycomprise an enzyme activity, or a primer. The enzyme activity may, forexample, be a DNA or RNA polymerase, such as an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specificribonuclease, or a DNA-dependent RNA polymerase.

In an embodiment of the invention, a lyophilisation formulation of theinvention comprises a polysaccharide, a low molecular weight saccharide,and enzyme activities required for self-sustained amplification of atarget nucleic acid (for example, using a method of the invention) inthe absence of enzyme cofactor(s) (for example magnesium ions), primers,rNTPs, and dNTPs required for specific amplification of the targetnucleic acid.

The enzyme activities may be RNA-dependent DNA polymerase, DNA-dependentDNA polymerase, DNA/RNA duplex-specific ribonuclease, and DNA-dependentRNA polymerase enzyme activities.

It will be appreciated that methods of the invention may be carried outin the presence of a polysaccharide and a low molecular weightsaccharide (for example, resulting from provision of reagents requiredfor amplification of the target nucleic acid with a lyophilisedformulation of the invention).

Examples of suitable polysaccharides are starch, a dextran, or aderivative of a dextran (for example dextran sulphate). The molecularweight of the polysaccharide is typically in the range from about10-2001 kD, usually 50-100 kD. The polysaccharide may be linear orbranched.

The low molecular weight saccharide may be a monosaccharide,disaccharide, or trisaccharide. Examples of suitable disaccharidesinclude trehalose, sucrose, and maltose. Rehydration speeds offormulations of the invention that include trehalose have been found tobe particularly fast. Inclusion of trehalose in lyophilised formulationsof the invention that comprise an enzyme has been found to maintain theactivity of the enzyme for long periods when stored at temperatures upto 37° C.

The low molecular weight saccharide may be present in an amount from2.5-15% (w/v) of the formulation.

The polysaccharide and low molecular weight saccharide may be present ina total amount of 4-12% (w/v) of the formulation.

A lyophilisation formulation of the invention may further comprise aninert protein such as BSA or casein. BSA can readily be obtained as anRNase-free preparation.

A lyophilisation formulation of the invention may further comprise asugar-alcohol.

There is also provided according to the invention a lyophilisationformulation of the invention which has been lyophilised (referred to asa lyophilised formulation of the invention). Methods of lyophilisationare known to those of ordinary skill in the art. A suitable method isdescribed in Example 3 below.

Lyophilised formulations of the invention can maintain labile reagentsin a stable condition at temperatures of 37° C. for at least a year(FIGS. 5 and 6 show that 100% signal strength is obtained after storageat 37° C. for one year). In contrast, lyophilised formulations describedin U.S. Pat. No. 5,556,771 show loss in activity after storage at 35° C.for 61 days (Table 4 of U.S. Pat. No. 5,556,771 shows that the averagerelative light units (RLU) for “Reagents with DNA target” at 61 days is90.9% of the average RLU at 0 days). The instructions provided with theSmartMix™ HM of Cepheid (a kit comprising lyophilised reagents for PCRamplifications) specifies that the lyophilised reagents must be storedat 2-8° C. The instructions provided with the commercially availableNuclisens® Basic Kit Amplification Reagents of Biomérieux (a kitcontaining lyophilised reagents for NASBA-based nucleic acidamplification) specify that the amplification reagents should be storedat ≤−20° C. The instructions provided with the commercially availableGEN-PROBE® APTIMA® General Purpose Reagents (GPR) 250 Kit (comprisinglyophilised reagents for carrying out transcription-mediatedamplification) specifies that the lyophilised reagents should be storedat 2-8° C. Lyophilised formulations of the invention do not need to berefrigerated.

The Applicant has surprisingly found that lyophilised formulations ofthe invention that comprise an enzyme (for example, an enzyme orcombination of enzymes required to carry out a method of the invention)are able to maintain the enzyme(s) in a stable condition even in theabsence of (or with little, e.g. <1 mM) buffering agent for theenzyme(s). This has the advantage that lyophilised enzyme formulationsare simplified. It may also be advantageous for other lyophilisedformulations comprising labile reagents besides enzymes to includelittle or no buffering agent.

Thus, according to the invention there is provided a lyophilised (orlyophilisation) formulation which comprises a polysaccharide, a lowmolecular weight saccharide and a labile reagent which it is desired topreserve in a stable condition, wherein the formulation does not includea buffering agent, or the buffering agent is present at a concentrationof less than 1 mM.

The term “buffering agent” is used to mean an agent that tends tomaintain a solution within a desired pH range (for example a pH range inwhich an enzyme retains activity). Typically, a weak acid or a weak baseis used. Examples of buffering agents used for transcription-basedamplification reactions include Tris-based buffers (e.g. Tris-HCl),HEPES, and acetate-based buffers. Phosphate buffering agents are notused for transcription-based amplification reactions because they areinhibitory to the amplification reaction.

According to the invention there is also provided a lyophilisedformulation for use in a method of the invention, comprising apolysaccharide, a low molecular weight saccharide, and a labile reagentrequired for carrying out a method of the invention which it is desiredto preserve in a stable condition, wherein the formulation does notinclude a buffering agent(s) required for the method or the bufferingagent is present at a concentration of less than that required for themethod. If buffering agent is present, it is preferred that this is at aconcentration of less than 25%, preferably less than 10%, of thatrequired for the method, or less than 1 mM.

The labile reagent may be an amplification reagent for amplifying thetarget nucleic acid or a detection reagent for detecting productresulting from amplification of the target nucleic acid.

In certain embodiments the labile reagent comprises an enzyme or enzymes(for example an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, a DNA/RNA duplex-specific ribonuclease, and a DNA-dependentRNA polymerase). Since the amount of enzyme substrate and any cofactorrequired for activity of the enzyme(s) may vary with differentprimer/template combinations, it is preferred that such formulations donot include substrates or cofactors for the enzyme or enzymes. It isalso preferred that such formulations do not include salt (for example,KCl, NaCl) since this again may vary with different primer/templatecombinations.

In other embodiments the labile reagent comprises primers for specificamplification of target nucleic acid using a method of the invention.Such formulations preferably further comprise nucleotides and/or anenzyme cofactor required for the method.

We have also found that lyophilised formulations of the invention can berapidly rehydrated without the need for a specialised reconstitutionbuffer. It has been found that known lyophilised formulations, forexample those described in U.S. Pat. No. 5,556,771, or commerciallyavailable lyophilised reagents (from BioMerieux, Gen-Probe, Cepheid), donot reconstitute rapidly, but instead require extensive mixing inspecially formulated reconstitution buffers. The instructions providedwith the Nuclisens® Basic Kit Amplification Reagents kit referred toabove specify that enzyme diluent should be added to the lyophilisedenzyme sphere, and left for at least 20 minutes at room temperature. Theinstructions provided with the SmartMix™ HM kit referred to abovespecify that the lyophilised beads are rehydrated after adding water byusing a vortex. In contrast, lyophilised formulations of the inventionreconstitute rapidly (usually in less than 10 seconds in water or otherliquid, such as nucleic acid extract) without any requirement forvortexing.

There is also provided according to the invention a kit comprising aplurality of different, separate lyophilised formulations of theinvention.

A kit of the invention may comprise reagents required for amplificationand/or detection of a target nucleic acid. The reagents may includeenzyme activities required for amplification of a target nucleic acid byPCR or by a self-sustained amplification reaction. A kit of theinvention may be for amplification of a target nucleic acid using amethod of the invention.

In some embodiments of kits of the invention which comprise enzymeactivities required for amplification of a target nucleic acid by aself-sustained amplification reaction, the enzyme activities may be in aseparate lyophilised formulation to the primers, ribonucleotidetriphosphates and deoxyribonucleotide triphosphates (and any enzymecofactors) required for specific amplification of the target nucleicacid.

A kit of the invention may comprise: i) a first lyophilised formulationof the invention, which includes an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease,and a DNA-dependent RNA polymerase; and ii) a second lyophilisedformulation of the invention, which includes primers for specificamplification of a target nucleic acid, ribonucleotide triphosphates forsynthesis of RNA by the DNA-dependent RNA polymerase, anddeoxyribonucleotide triphosphates for synthesis of DNA by the RNA- orDNA-dependent polymerase.

The RNA-dependent DNA polymerase and the DNA-dependent DNA polymerasemay be provided by a reverse transcriptase, such as reversetranscriptase from Avian Myeloblastosis Virus (AMV) or Moloney MurineLeukemia Virus (MMLV). The DNA/RNA duplex-specific ribonuclease may beprovided by RNase H, such as RNase H from E. coli or by the RNase Hactivity of AMV reverse transcriptase or MMLV reverse transcriptase. TheDNA-dependent RNA polymerase may be provided by T7 RNA polymerase. WhereAMV or MMLV reverse transcriptase is used, additional DNA/RNAduplex-specific ribonuclease activity may be provided by a separateenzyme (for example E. coli RNase H), as in conventional NASBA, or theactivity may be provided solely by the AMV-RT or MMLV-RT.

An enzyme cofactor(s) (such as magnesium ions) required by the enzymeactivities for amplification of the target nucleic acid may be providedas a separate component of the kit, or in the first or secondlyophilised formulation.

The Applicant has found that an enzyme stored in a lyophilisedformulation of the invention can remain stable for long periods attemperatures of 37° C. even if the formulation does not include anyreagents (for example, cofactors or substrates) required foramplification of a target nucleic acid using the enzyme. Often theoptimal mix of amplification reagents will vary with the target nucleicacid and the particular primers that are used. The applicant hasappreciated that if the amplification reagents are lyophilisedseparately from the enzymes, then a lyophilised enzyme formulation ofthe invention can be used with any target nucleic acid and primercombinations (i.e. as a universal lyophilised formulation). Since theamount of salt used may also vary with different target nucleic acid andprimer combinations, it may also be preferred to exclude salt from suchformulations.

Thus, the first lyophilised formulation may not include anyamplification reagents (for example, substrates or cofactors) requiredby the enzyme activities for amplification of the target nucleic acid.The first lyophilised formulation may also not include any salt.

The reverse transcriptase, DNA/RNA duplex-specific ribonuclease, andDNA-dependent RNA polymerase activities may each be provided by separateenzymes (although the reverse transcriptase may also include DNA/RNAduplex-specific ribonuclease activity, for example AMV-RT). This hasbeen found to reduce the complexity of the amplification mixturerequired to provide optimal enzyme activities at the temperature rangeof between 42 and 50° C. compared, for example, to use of enzymes (suchas MMLV reverse transcriptase) which comprise reverse transcriptase andRNase H activities (without an additional enzyme with DNA/RNAduplex-specific ribonuclease activity).

A kit of the invention for carrying out a method of the invention mayfurther comprise an agent for facilitating or enhancing theself-sustained amplification reaction, for example, any of the agentslisted above. The agent may form a separate component of the kit, or theagent may be in the first or second lyophilised formulation.

A kit of the invention for carrying out a method of the invention mayfurther comprise an agent that facilitates primer annealing, for examplepotassium or sodium ions. The agent may form a separate component of thekit, or the agent may be in the first or second lyophilised formulation.

A kit of the invention for carrying out a method of the invention mayfurther comprise an agent that facilitates denaturation of doublestranded nucleic acid during the amplification reaction, for exampleinosine triphosphate. The agent may form a separate component of thekit, or the agent may be in the first or second lyophilised formulation.

A kit of the invention for carrying out a method of the invention mayfurther comprise an agent that improves specificity of the amplificationreaction. The agent may form a separate component of the kit, or theagent may be in the first or second lyophilised formulation.

It may be desirable, however, that a polar aprotic solvent, such asDMSO, is not included as part of a lyophilisation (or lyophilised)formulation of the invention since this can adversely affectlyophilisation. As explained above, a zwitterionic compound, such asbetaine, may be used instead of DMSO since betaine does not appear toinhibit lyophilisation.

It has been found that high concentrations of a sugar-alcohol such asmannitol can hinder rehydration of a lyophilised formulation of theinvention. Consequently, it may be desired that if a sugar alcohol isincluded in a lyophilisation (or lyophilised) formulation of theinvention, it is present at less than 7.5% (w/v), less than 5% (w/v), orless than 3% (w/v). Alternatively, a sugar alcohol may be excluded froma formulation of the invention.

The first lyophilised formulation may further comprise an inert protein.

The second lyophilised formulation may comprise a polysaccharide, a lowmolecular weight saccharide, optionally a sugar alcohol, and any (orall) of the following: a reducing agent; positively charged ions; anenzyme cofactor (for example magnesium ions) required for activity ofthe amplification enzymes.

A kit of the invention may further comprise: iii) a third lyophilisedformulation of the invention, which includes a detection reagent fordetecting amplification product, and optionally iv) a fourth lyophilisedformulation of the invention, which includes a labelling reagent forlabelling the detection reagent.

In alternative embodiments, a kit of the invention may comprise: i) afirst lyophilised formulation of the invention, which includes adetection reagent for detecting target nucleic acid, or productresulting from amplification of target nucleic acid; and optionally ii)a second lyophilised formulation of the invention, which includes alabelling reagent for labelling the detection reagent.

The detection reagent may be any suitable reagent for detection ofamplification product or target nucleic acid. The detection reagent maycomprise a detection probe that hybridises to the amplification productor target nucleic acid. The detection reagent may itself be labelled(with one or more labels), thereby enabling direct detection of theamplification product or target nucleic acid utilising the detectionreagent. Alternatively, a labelling reagent (which comprises one or morelabels) for binding the detection reagent may be provided, therebyenabling indirect detection of the amplification product or targetnucleic acid utilising the detection and labelling reagents.

The label(s) of the detection reagent (where this is labelled) orlabelling reagent may be a visually detectable label. Examples ofvisually detectable labels include colloidal metal sol particles, latexparticles, or textile dye particles. An example of colloidal metal solparticles is colloidal gold particles.

The detection reagent may be a detection probe that is provided with aplurality of detection ligands (for example biotin), each of which canbe bound by a labelling reagent. Each labelling reagent may comprise aplurality of detection ligand binding moieties, each detection ligandbinding moiety being capable of binding a detection ligand of thedetection reagent. An example of such a labelling reagent is colloidalgold conjugated to antibiotin antibody. An example of the detectionprobe and labelling reagent is the detector probe and colouredanti-hapten detection conjugate, respectively, described and illustratedin Dineva et al (Journal of Clinical Microbiology, 2005, Vol. 43(8):4015-4021).

Detection of the amplification product may take place in standardhybridisation buffer. Examples of typical standard hybridisation buffersinclude a Tris or phosphate buffer comprising salt (suitably 100-400mM), surfactant (such as PVP), and a detergent.

It has been found that extracted nucleic acid in the elution buffer usedin conventional nucleic acid extraction procedures (usually a lowmolarity buffer, such as Tris, Tris-EDTA, Tris-HCl, or HEPES), or evenwater, provides a suitable sample solution for carrying out a method ofnucleic acid amplification according to the invention. Consequently, ifdesired a method of nucleic acid amplification of the invention can becarried out simply by contacting the enzyme activities required forself-sustained amplification of target nucleic acid, enzyme cofactor(s)required by the enzyme activities, primers required for specificamplification of target nucleic acid, required NTPs (rNTPs and dNTPs fortranscription-based amplifications), and (if appropriate) any of theagents listed above (for example by contacting first and secondlyophilised formulations provided in a kit of the invention), with theextracted nucleic acid in elution buffer (or water) and heating tobetween 42 and 50° C. Detection of amplification product (for example,as described above using a dipstick) can then be carried out simply bycontacting the detection reagents with the sample solution at the end ofthe amplification reaction, and contacting the resulting mixture with acontact end of the dipstick.

In an alternative aspect of the invention there is provided acomposition for use in a self-sustained amplification reaction (forexample, using a method of the invention), the composition comprisingenzyme activities required for self-sustained amplification of a targetnucleic acid (for example, using a method of the invention), and:

-   i) an agent for facilitating or enhancing the self-sustained    amplification reaction;-   ii) an agent that facilitates primer annealing;-   iii) an agent that facilitates denaturation of double stranded    nucleic acid; or-   iv) an agent that improves specificity of the self-sustained    amplification reaction.

The agents (i)-(iv) may be any of the agents listed above.

Combinations of agents may be used, such as: (i)+(ii); (i)+(iii);(i)+(iv); (ii)+(iii); (ii)+(iv); (iii)+(iv); (i)+(ii)+(iii);(i)+(ii)+(iv); or (i)+(iii)+(iv).

Use of such agents may improve the reliability, robustness (for exampleto temperature fluctuations), specificity and/or sensitivity of theamplification reaction. The Applicant has surprisingly found that use ofan inert protein, and an inert amphiphilic polymer (which is not aprotein) is particularly effective.

The composition may comprise enzyme activities required forself-sustained amplification of a target nucleic acid (for example usinga method of the invention), an inert protein, and an inert amphiphilicpolymer (which is not a protein). The composition may further comprisean agent that facilitates primer annealing and/or an agent thatfacilitates denaturation of double stranded nucleic acid and/or an agentthat improves specificity of an amplification reaction. The agent(s) maybe any of the agents listed above.

In a further aspect of the invention there is provided a composition forself-sustained amplification of a target nucleic acid (for example usinga method of the invention), which comprises: enzyme activities requiredfor self-sustained amplification of a target nucleic acid (for exampleusing a method of the invention); primers for specific amplification ofthe target nucleic acid; nucleotide triphosphates required for extensionof the primers (deoxyribonucleotide triphosphates and ribonucleotidetriphosphates are required for transcription-based amplification); andany of the following agents:

-   i) an agent for facilitating or enhancing the self-sustained    amplification reaction;-   ii) an agent that facilitates primer annealing;-   iii) an agent that facilitates denaturation of double stranded    nucleic acid; or-   iv) an agent that improves specificity of the self-sustained    amplification reaction.

The agents (i)-(iv) may be any of the agents listed above.

Again, combinations of agents may be used, such as: (i)+(ii); (i)+(iii);(i)+(iv); (ii)+(iii); (ii)+(iv); (iii)+(iv); (i)+(ii)+(iii);(i)+(ii)+(iv); or (i)+(iii)+(iv). Use of an inert protein, and an inertamphiphilic polymer (which is not a protein) is particularly effective.

The composition for self-sustained amplification of a target nucleicacid may comprise: enzyme activities required for self-sustainedamplification of the target nucleic acid; primers for specificamplification of the target nucleic acid; nucleotide triphosphatesrequired for extension of the primers (deoxyribonucleotide triphosphatesand ribonucleotide triphosphates are required for transcription-basedamplification); an inert protein; and an inert amphiphilic polymer(which is not a protein).

The composition may further comprise an agent that facilitates primerannealing and/or an agent that facilitates denaturation of doublestranded nucleic acid and/or an agent that improves specificity of anamplification reaction. The agent(s) may be any of the agents listedabove.

The enzyme activities of a composition of the invention may be: anRNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA/RNAduplex-specific ribonuclease, and a DNA-dependent RNA polymerase.

A composition of the invention may further comprise a buffer requiredfor the enzyme activities to carry out amplification of the targetnucleic acid in the presence of the primers, dNTPs, and rNTPs. Examplesof suitable buffers include Tris-HCl, HEPES, and acetate buffers.

A composition of the invention may further comprise any necessarycofactors required by the enzyme activities.

A composition of the invention may further comprise a low molecularweight saccharide and/or a sugar-alcohol. Alternatively, a compositionof the invention may further comprise a polysaccharide and a lowmolecular weight saccharide and/or a sugar alcohol. This may beparticularly suitable if the composition is lyophilised.

It will be appreciated that components of a composition of the inventioncan be provided in the form of a kit in which one or more components ofthe composition are separate from remaining components of thecomposition. Mixing of the separate components then provides acomposition of the invention.

The enzyme activities required for amplification of the target nucleicacid may be separate from the primers, NTPs, and any cofactors requiredby the enzyme activities.

Thus, there is also provided according to the invention a kit foramplification of a target nucleic acid by a self-sustained amplificationreaction (for example, carried out using a method of the invention),which comprises:

-   i) a first composition comprising enzyme activities required for    amplification of the target nucleic acid by the self-sustained    amplification reaction; and separately-   ii) a second composition comprising primers for specific    amplification of a target nucleic acid, NTPs required for extension    of the primers (ribonucleotide triphosphates and deoxyribonucleotide    triphosphates are required for transcription-based methods); and any    of the following agents:-   a) an agent for facilitating or enhancing the self-sustained    amplification reaction;-   b) an agent that facilitates primer annealing;-   c) an agent that facilitates denaturation of double stranded nucleic    acid;-   d) an agent that improves specificity of the self-sustained    amplification reaction.

The agent(s) may be a further separate component of the kit, or theagent(s) may be in the first or second composition.

The agents (a)-(d) may be any of the agents listed above. The firstcomposition may comprise an inert protein and/or the second compositionmay comprise a reducing agent and/or a sugar alcohol or low molecularweight saccharide.

The enzyme activities may be an RNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a DNA/RNA duplex-specific ribonuclease,and a DNA-dependent RNA polymerase.

The kit may further comprise a cofactor(s) required by the enzymeactivities. The cofactor(s) may be provided as a separate component ofthe kit or with the first or second composition.

A kit of the invention may further comprise an enzyme buffer requiredfor the enzyme activities to carry out amplification of the targetnucleic acid in the presence of the primers, dNTPs, and rNTPs. Examplesof suitable buffers include Tris-HCl, HEPES, and acetate buffers. Theenzyme buffer may be provided as a separate component of the kit or withthe first or second formulation or composition.

For those embodiments of the invention in which detection of theamplification product is carried out using a dipstick, a kit of theinvention may further comprise a dipstick capable of transporting asolution by capillarity.

A kit of the invention may further comprise instructions for carryingout a method of nucleic acid amplification and/or detection using thekit.

It may be desired to include with one or more separate components of akit of the invention a dye. For example, the separate components maycomprise different coloured dyes so that the different formulations orcompositions of the kit can readily be distinguished from each other.This can help reduce the risk of adding formulations or compositions tosample solution in the wrong order, or ensure correct preloading of adevice to carry out a method of the invention. Suitable dyes includefood dyes or textile dyes. These should be added at a concentration thatis not inhibitory to amplification or detection. For example,concentrations of 0.2% may be used.

It will generally be desirable to include with a formulation,composition, or kit of the invention an RNase inhibitor.

The components of a kit of the invention may be provided in a device forcarrying out a method of nucleic acid amplification and optionallydetection of nucleic acid product of the amplification reaction, thedevice being preloaded with the components.

Methods of the invention have been found to provide improved specificityof amplification of target nucleic acid, and reduced background,compared to conventional self-sustained amplification methods.

Methods of the invention are simpler and faster than conventionalmethods, and can be carried out without use of specialised labfacilities or instruments. We have also found that methods of theinvention are more tolerant to temperature fluctuations thanconventional NASBA methods (some methods of the invention are tolerantto temperature fluctuations of +/−4° C., compared with only +/−0.5° C.for conventional NASBA). Consequently, methods of the invention areparticularly suited for use in the field, in a physician's office, athome, in remote areas, or in developing countries where specialistfacilities and equipment may not be available.

Lyophilised formulations of the invention are stable for long periods atambient temperature and can be used to provide reagents required formethods of the invention. Because there is no need to store thelyophilised formulations at low temperatures, or to transport them bycold-chain transport, kits can be provided for carrying out methods ofthe invention in the field.

Reagents required for methods of the invention may be provided preloadedin a device comprising separate processing and analysing chambers thatare sealed from the external environment. The chambers are preferablyarranged so that processed sample (i.e. sample which has been incubatedunder conditions for amplification in the processing chamber) can bepassed into the analysing chamber without exposure to the externalenvironment. Such devices reduce contamination of the amplificationreaction, and thereby minimise the possibility of false positiveresults. This allows nucleic acid amplification reactions to be carriedout without the need for designated separate areas for samplepreparation, amplification, and detection, and facilitates use ofmethods of the invention in the field.

In certain aspects of the invention it may be desired to carry out aself-sustained nucleic acid amplification reaction at a temperature thatis lower than the range of between 42° C. and 50° C. (for example in therange 35-41° C.), but take advantage of other preferred aspects of theinvention. The preferred aspects may include, for example, any of thefollowing: use of one or more of the agents that facilitate or enhancethe self-sustained amplification reaction, or that facilitate primerannealing or denaturation of double stranded DNA, or that improve thespecificity of the amplification reaction; use of compositions orlyophilised formulations of the invention for the amplificationreaction; carrying out the amplification reaction in the presence of apolysaccharide and a low molecular weight saccharide; performing theamplification reaction in a processing chamber that is sealed from theexternal environment (and optionally detecting amplified product in ananalysing chamber sealed from the external environment); detectingamplified product using visually detectable labels.

There is also provided according to the invention a primer or probe thatcomprises or consists of a sequence recited in any of SEQ ID NOs: 1 to9.

The primers or probes of the invention may be up to 50, 40, or 30nucleotides in length.

There is further provided according to the invention a set of primersthat comprises a primer with sequence recited in SEQ ID NO: 1 and aprimer with sequence recited in SEQ ID NO: 2.

There is further provided according to the invention a set of primersand probes that comprises: a primer with sequence recited in SEQ ID NO:1; a primer with sequence recited in SEQ ID NO: 2; a probe with sequencerecited in SEQ ID NO: 3; a probe with sequence recited in SEQ ID NO: 4;and a probe with sequence recited in SEQ ID NO: 5.

The set may be used for amplification and/or detection of HIV-1 RNA.

There is further provided according to the invention a set of primersthat comprises a primer with sequence recited in SEQ ID NO: 6 and aprimer with sequence recited in SEQ ID NO: 7.

There is further provided according to the invention a set of primersand probes that comprises: a primer with sequence recited in SEQ ID NO:6; a primer with sequence recited in SEQ ID NO: 7; a probe with sequencerecited in SEQ ID NO: 8; and a probe with sequence recited in SEQ ID NO:9.

The set may be used for amplification and/or detection of Chlamydiatrachomatis RNA.

Embodiments of the invention are described in the following examples,with reference to the accompanying drawings in which:

FIG. 1 shows schematically the steps in transcription-basedamplification of a target RNA;

FIG. 2 illustrates steps of a method according to an embodiment of theinvention;

FIG. 3 shows the results of amplification and detection of HIV RNA usinga method according to an embodiment of the invention;

FIG. 4 shows the results of amplification and detection of Chlamydiatrachomatis RNA using a method according to a further embodiment of theinvention

FIG. 5 shows the results of amplification and detection of HIV RNA usinga method according to a further embodiment of the invention incomparison with results obtained from use of a commercially availableNASBA test;

FIG. 6 shows the results of a stability study carried out on lyophilisedformulations in accordance with a further embodiment of the invention;

FIG. 7 shows the results of a stability study carried out on lyophilisedformulations in accordance with a further embodiment of the invention;and

FIG. 8 shows the results of amplification and detection of Chlamydiatrachomatis RNA using a method according to an embodiment of theinvention in comparison with results obtained from use of a commerciallyavailable NASBA test.

EXAMPLE 1

a) Testing for HIV RNA

This example describes amplification and detection of HIV-1 RNA using anembodiment of the invention (referred to as Simple AMplificationBAsed-Nucleic Acid Testing (SAMBA-NAT)). The method steps are explainedbelow.

Lyophilised Formulations:

Lyophilised Bead A: 1 mM dNTP, 2 mM rATP, 2 mM rCTP, 2 mM rUTP, 1.5 mMrGTP, 0.5 mM rITP, 12 mM MgCl₂, 70 mM KCl, 5 mM DTT, 10 units RNaseinhibitor, 250 mM sorbitol, 1.5% dextran, 8.75% trehalose, and 0.2 mMprimers;

Lyophilised Bead B: 6.4 units AMV Reverse transcriptase, 0.16 unitsRNAse H, 32 units T7 RNA polymerase, 10 units RNase inhibitor, 1.5%(w/v) dextran, 8.75% (w/v) trehalose, 2 μg BSA;

Lyophilised Bead C: colloidal gold particles conjugated with anti-biotinantibody by passive absorption at a rate of 6 μg/OD(520 nm)/ml (referredto as “gold conjugate”), 1.5% dextran, 8.75% trehalose, 1% casein;

Lyophilised Bead D: 5×10¹¹ copies of detector probe (see below), 10¹²copies of helper probe (see below), 1.5% dextran, 8.75% trehalose.

Detection buffer: Standard Tris based hybridization buffer (pH 8.5)comprising salt, detergent and BSA or powdered milk with 0.05% sodiumazide.

Primers and Probes:

Sense primer ^(5′) CCT CAA TAA AGC TTG CCT TGA (SEQ ID NO: 1) andantisense primer ^(5′) GGC GCC ACT GCT AGA GA (SEQ ID NO: 2) elongatedat 5′-end with T7 promoter sequence.

Detector probe: ^(5′) CTC AAT AAA GCT TGC CTT GA (SEQ ID NO: 3) Captureprobe: ^(5′) CGT CTG TTG TGT GAC TCT GG (SEQ ID NO: 4) Helper probe:^(5′) GTG CTT CAA GTA GTG TGT GCC (SEQ ID NO: 5)

The detector and capture probes are prepared as described before (Dinevaet al, Journal of Clinical Microbiology, 2005, Vol. 43(8): 4015-4021).

DNA probes (20-25 nucleotides long) with sequence complementary tosequence of the target nucleic acid at a region adjacent to the regioncomplementary to the capture probe sequence could also be used aspreviously described in WO 02/04668.

Dipstick: a strip of nitrocellulose membrane comprising: a contact endfor contacting sample solution containing amplification product; and acapture zone for capturing amplification product. The capture probe isimmobilised to the capture zone as described by Dineva et al (Journal ofClinical Microbiology, 2005, Vol. 43(8): 4015-4021).

Method Steps:

-   Step 1: 200 ul of samples containing different amounts of HIV-1 RNA    (10⁵, 10⁴, 5×10³, 10³, 5×10², 2×10² copies/ml) (and a control    containing no HIV-1 RNA) were extracted using a Roche High Pure    Viral Nucleic Acid Kit or a Qiagen QIAamp Viral RNA Kit by following    instructions of the manufacturer.-   Step 2: 50 μl sample extract from step 1 was added to lyophilised    Bead A and mixed;-   Step 3: the solution from step 2 was incubated at 45° C. for 5    minutes;-   Step 4: lyophilised Bead B was added to the solution from step 3 and    mixed;-   Step 5: the solution from step 4 was incubated at 45° C. for 45    minutes;-   Step 6: 200 μl detection buffer was added to the solution from step    5;-   Step 7: lyophilised Beads C and D were added to the solution from    step 6 and mixed;-   Step 8: 100 μl of the solution from step 7 was contacted with the    contact end of the dipstick. The solution travels up the dipstick by    capillary action to a capture zone of the dipstick. If amplification    product is present in the solution, this is bound by the detector    probe provided with Bead D. The biotin moieties of the detector    probe are bound by the gold conjugate provided with Bead C.    Amplification product bound to the detector probe and gold conjugate    is captured at the capture zone by hybridisation of the    amplification product to the immobilised capture probe. Gold    conjugate bound to the biotin moieties of the detector probe    provides a visible signal at the capture zone if the amplification    product has been captured by the capture probe;-   Step 9: any signal appearing at the capture zone of the dipstick was    read.

The procedure from sample preparation to signal reading was completed in80 minutes. The results for each of the different samples are shown inFIG. 3, and demonstrate that as few as 200 copies of the RNA targetnucleic acid were detected using this procedure.

b) Testing for Chlamydia trachomatis RNA

The testing procedure for Chlamydia trachomatis is as shown in FIG. 2and similar to that for HIV RNA testing as described above with theexception of the primers and probes used, which have the following 16SrRNA specific sequences:

Sense primer ^(5′) AGC AAT TGT TTC GAC GAT TG (SEQ ID NO: 6) andantisense primer ^(5′) CA CAT AGA CTC TCC CTT AAC (SEQ ID NO: 7)elongated at 5′-end with T7 promoter sequence;

Detector probe: ^(5′) AAC TTG GGA ATA ACG GTT GGA A (SEQ ID NO: 8)Capture probe: ^(5′) CGC TAA TAC CGA ATG TGG CGA (SEQ ID NO: 9)

The results are shown in FIG. 4. As few as 50 copies of the targetnucleic acid were detected.

EXAMPLE 2

Comparison of Detection of HIV RNA Using SAMBA-NAT and NASBA-NAT

Detection of 10⁵, 10⁴, 10³, or 2×10² copies/ml of HIV RNA using aSAMBA-NAT method similar to that described in Example 1 was comparedwith amplification using a commercially available NASBA kit: NucliSensBasic Kit Amplification Reagents, Biomerieux (referred to as NASBA-NAT).For the NASBA-NAT this requires two additional incubation steps prior toaddition of enzyme mixture and amplification: a) incubation at 65° C.for 5 minutes, followed by b) incubation at 41° C. for 5 minutes.Amplicons prepared by either SAMBA-NAT or NASBA-NAT methods weredetected using an equivalent dipstick detection procedure to thatdescribed in Example 1. The results are shown in FIG. 5, and demonstratethat the SAMBA-NAT method detects HIV RNA with equivalent sensitivity tothe NASBA-NAT method. It can be seen from the zero (control) lane thatthe NASBA-NAT method provides a background signal. In contrast, nobackground signal is present in the SAMBA-NAT control lane. It isconcluded from this that the SAMBA-NAT method has higher specificity andreduced background interference compared to the NASBA-NAT method. TheSAMBA-NAT method was also faster than the NASBA-NAT method (45 minutescompared with 90 minutes).

The SAMBA amplification method described in this example has been foundto be more tolerant to temperature fluctuations than the conventionalNASBA amplification method. Conventional NASBA does not toleratetemperature fluctuations exceeding +/−0.5° C. of the optimalamplification temperature of 41° C. In contrast, the SAMBA method istolerant to temperature fluctuations of +/−4° C.

Table 1 below summarises some of the main advantages of the SAMBAamplification method over the conventional NASBA amplification method.

TABLE 1 NASBA Disadvantage SAMBA Advantage Sensitive to temperaturefluctuations Tolerant to temperature exceeding +/−0.5° C. fluctuationsof +/−4° C. Three incubation steps and at least two Single incubationtemperature different incubation temperatures Reagents required to bestored at −20° C. Reagents stable above 30° C. and transported bycold-chain transport More than 10 individually packed reagents Only twolyophilised required formulations for amplification Amplification timeis approximately 90 Amplification time is minutes approximately 45minutes

EXAMPLE 3

Long-term Stability of Enzymes in Lyophilised Formulations

An aqueous solution comprising 1.5% dextran, 8.75% trehalose, 6.4 U AMVReverse transcriptase, 0.16 U RNAse H, 32 U of T7 RNA polymerase and 2μg BSA was prepared and then split into several aliquots, each of 25 μl.Each aliquot was lyophilised by dispensing the aqueous solution intocryogenic liquid nitrogen. The solution freezes as discrete sphericalparticles upon contact with the cryogenic agent. The frozen particlesare further subjected to a vacuum while still frozen under pressure(˜0.1 mbar) and for time (20-48 hours) sufficient to remove the solventusing freeze drier machine (Christ Alpha 2-4, Martin Christ GmbH,Osterode am Harz, Germany). The lyophilised aliquots were then stored at−20° C., 4° C., 25° C., or 37° C. Lyophilised aliquots stored at each ofthe different temperatures were tested after intervals of 10 days, 1month, 2 months, 3 months, 9 months, and 12 months to determine thestability of the enzymes in the lyophilised aliquots. Stability wastested by carrying out amplification of a nucleic acid target, anddetection of the amplified product using a method similar to thatdescribed in Example 1. The detection signal obtained from each aliquotwas compared to that obtained using a freshly made up sample. Theresults are shown in FIG. 6, and demonstrate that the enzymes remainedactive after being stored for 12 months at temperatures up to 37° C.

EXAMPLE 4

Long-term Stability of Other Labile Reagents in Lyophilised Formulations

An aqueous solution comprising 2 mM dNTP, 4 mM rATP, 4 mM rCTP, 4 mMrUTP, 3 mM rGTP, 1 mM rITP, 24 mM MgCl₂, 140 mM KCl, 10 mM DTT, 10 unitsRNase inhibitor, 500 mM sorbitol, 1.5% dextran, 8.75% trehalose, andprimers was prepared and then split into several aliquots, each of 25μl. Each aliquot was lyophilised by the method as described in Example3. The lyophilised aliquots were then stored at −20° C., 4° C., 25° C.,or 37° C. Lyophilised aliquots stored at each of the differenttemperatures were tested after intervals of 10 days, 1 month, 2 months,3 months, 9 months, and 12 months to determine the stability of thereagents in the lyophilised aliquots. Stability was tested by carryingout amplification of a nucleic acid target, and detection of theamplified product using a method similar to that described in Example 1.The detection signal obtained from each aliquot was compared to thatobtained using a freshly made up sample. The results are shown in FIG.7, and demonstrate that the reagents remained stable after being storedfor 12 months at temperatures up to 37° C.

EXAMPLE 5

Rehydration Speed of Lyophilised Formulations

Aqueous solutions were made up (final volume 25 μl) containing dextran,and either sucrose, trehalose, mannitol, or trehalose and mannitol (atvarious different concentrations). The solutions were then lyophilisedusing a similar method as that described in Example 3. Water (50 μl) wasadded to each lyophilised formulation in turn, and the tube containingthe formulation was flicked with a finger until rehydration hadoccurred. The time taken for rehydration (number of flicks, ˜1flick/second) was recorded. The results are shown in Table 2 below.

The results demonstrate that the fastest rehydration times are obtainedwith lyophilised formulations comprising dextran and trehalose, althoughrehydration times with dextran and sucrose are nearly as fast. Presenceof higher concentrations of mannitol (i.e. 7.5 mM or higher) appears toinhibit rehydration times.

TABLE 2 Final concentration Rehydration time Formulation Reagents (mM)(arbitrary units) A Dextran 1.5 3-4 Sucrose 7.5 B Dextran 1.5 4 Sucrose8.75 C Dextran 1.5 5-6 Sucrose 10.5 D Dextran 1.5 3 Trehalose 7.5 EDextran 1.5 3 Trehalose 8.75 F Dextran 1.5 3-4 Trehalose 10.5 G Dextran1.5 18-20 Mannitol 7.5 H Dextran 1.5 20 Mannitol 8.75 I Dextran 1.526-27 Mannitol 10.5 J Dextran 1.5 3-4 Mannitol 2.5 Trehalose 8.75 KDextran 1.5 16-17 Mannitol 7.5 Trehalose 8.75

EXAMPLE 6

Accelerated Stability Study of Enzymes in Lyophilised Formulations

Aliquots of lyophilised enzymes prepared as described in Example 3 werestored at 25° C., 37° C. or 55° C. Lyophilised aliquots stored at eachof the different temperatures were tested as described in Example 3after intervals of 3, 7, 14, 21 days and 1 month to determine thestability of the enzymes in the lyophilised aliquots at elevatedtemperature. The results obtained demonstrate that the enzymes remainedactive after being stored for 1 month even at 55° C.

EXAMPLE 7

Comparison of Detection of Chlamydia trachomatis 16S rRNA Using ofSAMBA-NAT and Conventional NASBA

Detection of 10⁴, 2×10³, 5×10², 2×10², 75 or 10 copies/test of Chlamydiatrachomatis 16S rRNA using the SAMBA-NAT method as described in Example1(b) was compared with detection using conventional NASBA (whichrequires two additional incubation steps prior to addition of enzymemixture and amplification: a) incubation at 65° C. for 5 minutes,followed by b) 5 minutes cooling at 41° C.). Amplicons prepared byeither SAMBA-NAT or conventional NASBA methods were detected using anequivalent dipstick detection procedure to that described in Example 1.The results are shown in Table 3 below, and FIG. 8, and demonstrate thatthe SAMBA amplification protocol detects Chlamydia trachomatis 16S rRNAwith more than 7.5 times increased detection sensitivity than the morecomplex conventional NASBA method.

TABLE 3 Copies/test of Chlamydia trachomatis 16S rRNA SAMBA NASBA 100005 5 2000 5 5 500 5 4 200 5 3.5 75 5 1.5 10 5 0

The invention claimed is:
 1. A universal lyophilised formulationcomprising a polysaccharide, a low molecular weight saccharide, andenzyme activities required for carrying out a self-sustainedamplification reaction, wherein the enzyme activities are provided byone or more enzymes, and wherein the formulation does not includecofactors or substrates required for amplification of a target nucleicacid using the enzyme activities.
 2. A formulation according to claim 1,which does not include salt.
 3. A formulation according to claim 1 or 2,wherein the polysaccharide is a dextran or a dextran derivative.
 4. Aformulation according to any of claims 1 to 3, wherein the low molecularweight saccharide is trehalose, sucrose, or maltose.
 5. A lyophilisedformulation comprising a polysaccharide, a low molecular weightsaccharide, and enzyme activities required for carrying out aself-sustained amplification reaction, wherein the enzyme activities areprovided by one or more enzymes, and wherein the formulation does notinclude a buffering agent.
 6. A formulation according to claim 5,wherein the polysaccharide is a dextran or a dextran derivative.
 7. Aformulation according to claim 5 or 6, wherein the low molecular weightsaccharide is trehalose, sucrose, or maltose.
 8. A formulation accordingto claim 5, wherein the enzyme activities comprise an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specificribonuclease, and a DNA-dependent RNA polymerase.
 9. A formulationaccording to claim 5, which does not include substrates or cofactors forthe enzyme activities.
 10. A formulation according to claim 9, whichdoes not include salt.
 11. A kit comprising a plurality of different,separate lyophilised formulations wherein each lyophilised formulationis a lyophilised formulation according to claim 1 or
 5. 12. A universallyophilised formulation comprising a polysaccharide, a low molecularweight saccharide, and enzyme activities required for carrying out aself-sustained amplification reaction, wherein the enzyme activities areprovided by one or more enzymes, and wherein the formulation does notinclude cofactors or substrates required for amplification of a targetnucleic acid using the enzyme activities, and wherein the formulationdoes not include a buffering agent, or the buffering agent is present ata concentration of less than 1 mM.
 13. A universal lyophilizedformulation according to claim 1, wherein the enzyme activities comprisean RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA/RNA duplex-specific ribonuclease, and a DNA-dependent RNApolymerase.
 14. A universal lyophilized formulation according to claim12, wherein the enzyme activities comprise an RNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a DNA/RNA duplex-specificribonuclease, and a DNA-dependent RNA polymerase.